从研究职业发展奖防盲支持，国立卫生研究院资助EY19312 EY19543（VJK），以及不受限制地从研究的拨款，以防止盲目性和EY02687（华盛顿大学眼科及视觉科学系）。

1。使电极<ol><li>玻璃电极的准备。重达120毫克，琼脂和混合在10毫升蒸馏水（最终琼脂浓度为1.2％）。在热水浴融化的琼脂溶液。填补玻璃毛细管（我们使用以下尺寸的Word Presision仪器TW100-4毛细血管长度为100毫米，外径/ ID = 1/0.75毫米，内部容积为44μL）琼脂解决方案，使用塑料注射器。巩固在室温下10分钟的琼脂。因此，最多可以填写200毛细血管（琼脂混合物的其余部分可以存放在-4°C和重新使用几次）。 </li><li>使用钻石刀切成一半的毛细血管。 </li><li>浸泡24小时，至少在4°C的玻璃电极在导致鼠标电极解决方案储存在4°C的电极，并在3-4个月内使用它们。 </li></ol> 2。设立实验<ol><li>一夜之间暗适应鼠标。使用野生型（例如，C57Bl / 6）小鼠控制棒驱动transretinal的录音和Tα - / -小鼠缺乏杆锥transretinal录音信号。 </li><li>准备1升灌注液，100毫升的电极解决方案，并培养20毫升的解决方案（见下文）和过滤灌注液槽1 Millipore公司0.45微米或0.22微米过滤器（可选）。 </li><li>使用放置在视网膜平面光度计校准光源（505 nm的LED或卤素灯泡的光刺激）。在LED光源的情况下，使用光学扩散，达到光线均匀。 </li><li>准备灌注室实验（室结构可能会有所不同）。我们室的3毫米有机玻璃制成，从一个小塑料培养皿的盖子底部，有一个积极的〜灌注量400μL（L为25毫米，宽8毫米，H≈2毫米）。填充电极的含L-谷氨酸2毫米和10毫米氯化钡2解决方案下电极室空间。用塑料管材和玻璃毛细管，在较低的电极空间（室底部的塑料管材连接）和电极支架的解决方案之间的连接。确保无气泡在电极，连接管，或电极支架（清除解决方案，如果需要使用塑料注射器）。 </li><li>安装在显微镜的阶段（可选：显微镜可以安装在防振表，并在一个法拉第笼屏蔽，以减少机械和电磁噪音）的录音室。较低的电极支架连接到差分放大器（如华纳仪器DP-311放大器）正探头极。中心和校准室较低的电极刺激光斑开放。灌注线连接到录音室，开启灌注。 </li><li>泡沫灌注液与95％的O 2/5％CO 2，同时孵化，在40°çWAT呃浴。 CO 2调整的解决方案，以7.2的pH值。解决方案的流向从瓶子到录音室，通过陶瓷电阻，通过重力将它加热到36-37°C。为了减少热量损失，热水器应设为尽可能接近录音室，最好在显微镜阶段。 </li><li>随着流量调节器调节流速约1毫升/分钟。 </li><li>上电极连接使用第二个电极支架的探头第二（负）极。热电偶探头结合使用O形圈的上部电极。确保电极和热电偶提示并拢尽可能接近视网膜的中心，使温度读数。上电极浸入灌注液中的一角，放置在室中心。允许基线稳定10-15分钟。 </li><li>直流电压调整加热器，使溶液的温度是36 -37°C。 </li></ol> 3。隔离小鼠视网膜<ol><li>在昏暗的红灯，安乐死与CO 2和颈椎脱位，暗适应的鼠标。可选：有烧灼笔或加热的解剖针烧灼巩膜最背点，通过对每个鼠标眼球的参考标记。所有后续的程序进行，使用红外图像转换器，适合在解剖显微镜下红外光。 </li><li>解剖施加温和的压力和伸展每只眼睛的两侧皮肤弯剪刀的眼睛。 Hemisect下红外照明使用microscissors或刀片的两个眼球。 </li><li>取下的角膜和晶状体。删除尽可能多的玻璃体。 </li><li>从第一视网膜色素上皮细胞层和剥离，如果必要的话，彻底清洁，用钳子消除色素上皮剩余的颗粒。我们通常使用为整个视网膜杆锥transret都inal录音。不过，如果你想专门记录才剥离视网膜删除眼罩的腹侧部分，用刀片或烧灼标志作为参考使用microscissors，从背小鼠视网膜的一部分，有更多的M / L型锥体9。 </li><li>将Petri网与孵化的解决方案中的一个小盘子的第二个半横断眼球，直到使用纯氧饱和光紧盒中孵化的准备。通常，在这些条件下第二鼠标眼罩可以被储存在室温下几个小时的录音使用。 </li><li>使用玻璃或塑料吸管孤立的视网膜转移到培养皿的盖子上的灌注液中（约300μL）。新增5-6电极溶液氯化钡2预孵育的视网膜含有氯化钡2的小水珠。将背面的方形片（约5×5毫米）角脂（我们使用道康宁111阀门润滑剂和密封剂）微孔滤纸（0.45微米HARG型），在其中心的预制孔（直径2-2.5毫米）相同的解决方案降。使用镊子位置滤纸的顶部（感光朝上）视网膜和按平面安装的外围边缘。应集中在视网膜在滤纸开幕。 </li><li>使用镊子转移滤纸与视网膜灌注室和凌驾于刺激点对准下电极空间。稍微按脂滤纸的边缘，债券，同室。 </li><li>放置在视网膜中心（略触摸感光层）上电极。围绕视网膜上的电极，提高了信号的幅度（高达20-40％，而把它接近视网膜的边缘）。虽然这种电极的位置稍微歪曲的有效光刺激强度达到视网膜（其总面积的12-15％）的小部门，它仍然是放置电极的最佳方式电极。准备准备现在的transretinal记录。 </li></ol> 4。 transretinal录音<ol><li>根据你的鼠标线，纪录杆或M / L型锥驱动的测试闪光反应，从朦胧到明亮的校准505 nm的光从LED光源或光刺激强度。在我们的LED光源的情况下，闪光强度是由一个电脑控制的LED输入电压，切换电阻，和一套中性密度滤镜（我们用E-色＃211 0.9 ND ROSCO实验室的膜过滤器的组合控制）LED和视网膜之间。由计算机控制的LED测试闪光的持续时间（20毫秒）。如果使用卤素灯泡的光刺激，测试闪光强度和波长可以由一组中性密度过滤器和窄带干涉滤光片，分别控制。测试闪光持续时间可以控制由计算机驱动的百叶窗。 </li><li>可选：为保持高效率全天光响应与氯化钡2胶质成分的抑制，偶尔改变下电极的解决方案（例如之前测试每个视网膜）通过清除它充满了新鲜的溶液用塑料注射器。 </li></ol> 5。代表结果解决方案<ol><li>小鼠视网膜的灌注液：氯化钠112.5毫米，3.6毫米，2.4毫米氯化镁氯化钾， 氯化钙 1.2毫米，10毫米的肝素钠（pH值7.2）， 碳酸氢钠 20毫米，3毫米钠琥珀酸，谷氨酸钠0.5毫米，0.02毫米EDTA，和10 mM葡萄糖。此外，该解决方案的补充，与2毫米L-谷氨酸和10μM的DL - AP-4，以阻止高阶元件的光响应10,11和0.1％的MEM维生素和MEM氨基酸的解决方案（Sigma公司），以改善视网膜生存能力。 </li><li>鼠标电极解决方案：140毫米3.6毫米2.4毫米1.2毫米，3毫米肝素钠（pH值7.4，氢氧化钠） 氯化钙氯化镁 ，氯化钾，氯化钠，。在较低的元素的解决方案ctrode空间还包含2毫米L-谷氨酸阻断突触传递10，此外，10毫米氯化钡2抑制胶质细胞的光响应3,12组成部分。 </li><li>视网膜孵化溶液：溶解272毫克的L-15培养基（Sigma公司）和20 mg BSA在20 mL去离子水。最后的L-15和BSA浓度为13.6毫克/毫升和1毫克/毫升，分别。 1 M盐酸调整pH值至7.4。 </li></ol><img alt="图1。" src="/files/ftp_upload/3424/3424fig1.jpg" /> 图1。示意图隔离的小鼠视网膜为transretinal ERG的记录。 <img alt="图2。" src="/files/ftp_upload/3424/3424fig2.jpg" /> 图2。灌注室和记录电极与持有人的照片。红色箭头指示方向的灌注流量。 <img alt="图3。" src="/files/ftp_upload/3424/3424fig3.jpg"/&gt; 图3。的为transretinal视网膜录音实验装置示意图。红色箭头指示方向的灌注流量。 <img alt="图4。" src="/files/ftp_upload/3424/3424fig4.jpg" /> 图4代表结果：记录家庭鼠标杆驱动transretinal反应。光反应是低通滤波（8极贝塞尔）在30 Hz，1 kHz时的数字化，并存储在计算机作进一步的分析。所示的痕迹，在昏暗的光线强度和饱和光照强度2-3答复代表平均5-6答复。 <img alt="图5。" src="/files/ftp_upload/3424/3424fig5.jpg" /> 图5代表结果：记录家庭的鼠标驱动锥transretinal反应。光反应是低通滤波（8极贝塞尔）在30 Hz，1 kHz时的数字化，并存储在计算机作进一步的分析。所示的痕迹是求均值5-10反应ES在昏暗的光线强度和饱和光照强度3-5反应。

<ol>
	<li>Yau, K. W., Lamb, T. D., Baylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+fluctuations+in+membrane+current+of+single+toad+rod+outer+segments.">Light-induced fluctuations in membrane current of single toad rod outer segments.</a> Nature. 269, 78-80 (1977).</li><li>Nikonov, S. S., Kholodenko, R., Lem, J., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+features+of+the+S-+and+M-cone+photoreceptors+of+wild-type+mice+from+single-cell+recordings.">Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.</a> J. Gen. Physiol. 127, 359-374 (2006).</li><li>Nymark, S., Heikkinen, H., Haldin, C., Donner, K., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+responses+and+light+adaptation+in+rat+retinal+rods+at+different+temperatures.">Light responses and light adaptation in rat retinal rods at different temperatures.</a> J. Physiol. 567, 923-938 (2005).</li><li>Heikkinen, H., Nymark, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cone+photoresponses+obtained+with+electroretinogram+from+the+isolated+retina.">Mouse cone photoresponses obtained with electroretinogram from the isolated retina.</a> Vision Res. 48, 264-272 (2008).</li><li>Frank, R. N., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+photoproducts:+effects+on+electroretinogram+sensitivity+in+isolated+perfused+rat+retina.">Rhodopsin photoproducts: effects on electroretinogram sensitivity in isolated perfused rat retina.</a> Science. 161, 487-489 (1968).</li><li>Wang, J. S., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+pathway+mediates+the+mouse+and+human+cone+visual+cycle.">An alternative pathway mediates the mouse and human cone visual cycle.</a> Curr. Biol. 19, 1665-1669 (2009).</li><li>Kolesnikov, A. V., Tang, P. H., Parker, R. O., Crouch, R. K., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+cone+visual+cycle+promotes+rapid+M/L-cone+pigment+regeneration+independently+of+the+interphotoreceptor+retinoid-binding+protein.">The mammalian cone visual cycle promotes rapid M/L-cone pigment regeneration independently of the interphotoreceptor retinoid-binding protein.</a> J. Neurosci. 31, (2011).</li><li>Calvert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototransduction+in+transgenic+mice+after+targeted+deletion+of+the+rod+transducin+alpha+-subunit.">Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 13913-13918 (2000).</li><li>Applebury, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+cone+photoreceptor:+a+single+cone+type+expresses+both+S+and+M+opsins+with+retinal+spatial+patterning.">The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.</a> Neuron. 27, 513-523 (2000).</li><li>Sillman, A. J., Ito, H., Tomita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mass+receptor+potential+of+the+isolated+frog+retina.+I.+General+properties+of+the+response.">Studies on the mass receptor potential of the isolated frog retina. I. General properties of the response.</a> Vision Res. 9, 1435-1442 (1969).</li><li>Vinberg, F., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sets+the+physiological+value+of+the+dominant+time+constant+of+saturated+mouse+rod+photoresponse+recovery.">Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery.</a> PLoS One. 5, e13025-e13025 (2010).</li><li>Bolnick, D. A., Walter, A. E., Sillman, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barium+suppresses+slow+PIII+in+perfused+bullfrog+retina.">Barium suppresses slow PIII in perfused bullfrog retina.</a> Vision Res. 19, 1117-1119 (1979).</li></ol>

作者没有透露。

杆和锥驱动transretinal ERG的录音上面介绍的方法，正在成为一个调查，在野生型和转基因动物的鼠标感光功能强大的工具。除了基本的光响应特性的简单特征，这个简单的技术提供了很大的反响稳定性，在近乎完整的视网膜准备进行长期的实验。无论是暗适应的棒最大响应幅度和野生型小鼠光敏性至少1小时内是稳定的，从开始录音，暗适应锥最大振幅和光敏性通常不下降30-40分钟后超过10％录音。?...

<table border="1"><tbody><tr><td> 材料名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DL - AP-4 </td><td></td><td> TOCRIS生物科学</td><td> 0101 </td><td></td></tr><tr><td>所有其他试剂</td><td></td><td> Sigma-Aldrich公司</td><td></td><td></td></tr><tr><td>玻璃毛细管</td><td></td><td>世界精密仪器</td><td> TW100-4 </td><td>为了使电极</td></tr><tr><td>滤纸</td><td> HARG </td><td> Millipore公司</td><td> HABG01300 </td><td></td></tr><tr><td>光度计</td><td></td><td> UDT的仪器</td><td> S350的</td><td>对于光校准</td></tr><tr><td>辐射硅传感器</td><td></td&gt; <td> UDT的仪器</td><td> 221 </td><td>对于光校准</td></tr><tr><td>抗振动表</td><td></td><td>技术制造公司</td><td>丰田汽车公司78-239-02R，TMC63-26012-01 </td><td>为了尽量减少机械噪音</td></tr><tr><td>空气压缩机豹带够15TC </td><td></td><td>维特国际</td><td>带够15TC </td><td>与防振表连接到</td></tr><tr><td>体视显微镜</td><td></td><td>徕卡</td><td> MZ9.5 </td><td>鼠标眼清扫</td></tr><tr><td>红外图像转换器</td><td></td><td> BEMeyers </td><td> ProwlerTM </td><td>绑定到立体显微镜</td></tr><tr><td>差分放大器 DP-311 </td><td></td><td>华纳仪器</td><td> DP-311 </td><td></td></tr><tr><td>轴突Digidata 1440A数字化仪</td><td>; </td><td>分子器件</td><td> 1440A </td><td></td></tr><tr><td>双通道8极过滤器</td><td></td><td>克罗恩-海特公司</td><td> 3382 </td><td></td></tr><tr><td>陶瓷电阻</td><td></td><td> TE的连通</td><td>总参谋长SBCHE618RJ </td><td>加热灌注液</td></tr><tr><td>热电偶</td><td> ţ </td><td> physitemp仪器</td><td> IT-18 </td><td></td></tr><tr><td>温度监视器</td><td></td><td>欧米茄</td><td> DPi32 </td><td>连接到热电偶</td></tr><tr><td> LED的505纳米</td><td></td><td> TT电子/ OPTEK技术</td><td>从DIGI-KEY的P / N 365-1185-ND </td><td>申请测试闪烁/漂白光</td></tr><tr><td>烧灼笔</td><td></td><td> bovie </td><td> AA25 </td><td>为标志的背侧鼠标眼球</td></tr><tr><td> pCLAMP 10电数据采集和分析软件</td><td></td><td>分子器件</td><td></td><td></td></tr></tbody></table>

transretinal erg recordings from mouse retina: rod and cone photoresponses

有两个不同的类脊椎动物的视网膜成像感光杆和视锥细胞。棒是能够探测到单光子的光，而视锥细胞在瞬息万变的明亮的光线条件下连续运行。光杆和锥感光细胞的外节的视觉色素的吸收触发一个光传导级联反应，最终导致封闭的环核苷酸门控通道的细胞膜和细胞极化。现有的和潜在的这种膜的光致变化可以注册为光响应，无论是古典吸电极记录技术1,2或transretinal药物阻断突触后反应元件3-5孤立视网膜电图记录，从图（ERG）。后一种方法允许从小鼠感光细胞的药物访问长久的录音和获得稳定的光反应FR特别有用OM的稀缺和脆弱的鼠标锥。在视锥细胞的情况下，进行这样的试验，无论是在暗适应条件和以下强烈的光照漂白基本上所有的视觉色素，监察锥感光恢复的过程中，在暗适应6,7。在这部影片中，我们将展示如何进行暗适应小鼠视网膜棒和M / L，锥的驱动transretinal录音。使用野生型小鼠（C57BL / 6）视网膜棒的录音将进行。为简单起见，锥录音将获得杆转导的转基因α-亚基基因敲除（Tα - / - ）小鼠缺乏信号8杆。

Watch this Scientific Journal Video about Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses at JoVE.com

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

我们描述一个transretinal视网膜电图（ERG）录音获得完整的小鼠视网膜的杆状和锥状光反应相对简单的方法。这种做法从感光细胞的突触传递块孤立的光反应，并记录他们使用场电极置于整个平板式的孤立视网膜。

transretinal ERG的录音，从小鼠视网膜杆和锥光反应

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

transretinal-erg-recordings-from-mouse-retina-rod-cone

Research

JoVE Journal

Neuroscience

17.6K 次观看.  Washington University School of Medicine. 我们描述一个transretinal视网膜电图（ERG）录音获得完整的小鼠视网膜的杆状和锥状光反应相对简单的方法。这种做法从感光细胞的突触传递块孤立的光反应，并记录他们使用场电极置于整个平板式的孤立视网膜。

视频: Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

的vibratome切片小鼠视网膜准备感光文化

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

你在哪里切的问题: 眼地标性小鼠视网膜空间定位的解剖与分析指南

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

自由移动仔猪的无创脑电图记录

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Visualizing Axonal Growth Cone Collapse and Early Amyloid &amp;#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

在培养的小鼠神经元中可视化轴突生长锥塌陷和早期淀粉样β效应

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

从鸡胚胎的直膜到研究棒到锥细胞相互作用的锥形丰富文化

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

体内 小鼠视网膜血管损伤读数以促进可重复性

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Mouse Retina Isolation and Flattening

After fixing the enucleated eyeball of the euthanized mouse, transfer it to a glass slide and place it under a dissecting microscope. Using forceps, clamp the margin of the cornea to prevent eyeball movement. With the dissecting scissors, cut around the cornea at a depth of approximately one millimeter through the cornea scleral limbus, and then remove the lens and vitreous body.Holding the optic disc at the center, cut the retina radially along the four quadrants, reaching approximately 2/3 of the retina's radius. Clamp one of the scleral edges using toothed forceps. Hold the sclera edge with untoothed forceps and move from the toothed forceps toward the sclera base.Carefully peel the retina. Wet the retina with 200 microliters of PBS using a pipette. Remove excess PBS using absorbent paper, and flatten the retina with a small bristle brush if needed.Slowly add cold methanol drop-by-drop onto the retina's inner surface until it covers completely. Once the retina turns white and develops increased tenacity, gently flatten the retina and remove impurities, such as residual pigment membranes and the vitreous body, with a small bristle brush. After inverting the retina, treat it with methanol as previously demonstrated, and transfer it into a well of 24 well plate, Soak it in methanol and store it at 20 degrees Celsius for one to two hours for immediate immunostaining.Then rinse the retina with PBS after removing methanol from the plate. The retinol ganglion cells of the retinas before and after methanol treatment were visualized using confocal microscopy, which indicated that the 12 months of methanol preservation did not affect the RGC immunostaining.